Immobilized Aspergillus oryzae protease (AOP) catalyzed the formation of peptide bonds between TV‐protected amino acids and amino acid esters or amides in ethyl acetate. The influences of pH and reaction time on the coupling of Boc‐L‐Tyr and Gly‐NH2 were studied. The optimal reaction condition for this enzyme catalyzed synthesis of Boc‐L‐Phe‐Gly‐NH2 (98.66%) was at pH 5.5 and a duration of 48 hours.